Project Summary: Glaucoma is a leading cause of irreversible blindness in the human population. Glaucoma is frequently associated with elevated intraocular pressure (IOP), but IOP alone is neither predictive of vision loss, nor the sole risk factor for disease progression, and its tendency to fluctuate over time makes it a suboptimal screening tool and prognostic indicator. The structural and biomechanical properties of the extracellular matrix, and in particular the collagen that makes up the sclera, have been widely implicated as playing a significant role in glaucoma susceptibility, though the precise nature of this relationship remains unresolved. Given the recognized influence of scleral properties on both IOP profile and optic nerve head deformation and damage in glaucoma, there is a critical need for non-invasive, cost-effective methods to quantify collagen microstructure in the sclera. Detection and quantification of optical scattering is sensitive to structural organization of tissue down to the nanoscale, well below the diffraction limited resolution of other imaging methods. Enhanced Backscattering (EBS) has been applied in a clinical setting to screen for cancer risk. We hypothesize that glaucoma-associated differences in scleral collagen structure can be quantified using EBS. However, EBS has not previously been applied to fibrous tissues like sclera, and assessment of scleral collagen structure will require development of new tissue-specific EBS approaches and hardware as well as new models for analyses. To overcome this barrier to clearer understanding of the role played by scleral collagen structure in glaucoma pathobiology, with the ultimate goal of developing a tool with translational potential, we propose two specific aims. In Aim 1: we will build the first polarimetric EBS instrument specifically optimized for sclera. This novel instrument will incorporate angular bandwidth matched to higher scattering by sclera and polarization control to provide sensitivity to birefringence of this fibrous tissue. The instrument will be validated using precision microsphere phantoms and applied in a unique animal model of glaucoma with acquisition of measurements from normal and glaucomatous sclera specimens ex vivo. In Aim 2: We will develop a computational model to relate EBS measurements to tissue collagen structure as determined by second harmonic generation microscopy, and based on scattering phase function from sclera using light scattering goniometry. The most significant differences in optical scattering signature between groups will be identified as a promising biomarker. Together, these experiments will provide a novel instrument and model with potential for translation to pre-clinical and clinical settings. In particular, the non-invasive nature of the test will permit much needed longitudinal studies of changes in collagen properties within individuals with spontaneous glaucoma, and also changes in response to age and interventions.